At present, preparations comprising granulocyte colony-stimulating factor (G-CSF) are used against disorders and clinical conditions accompanied by reduction of neutrophils. These preparations may be administered by intravenous injection, subcutaneous injection, or drip infusion, but they should be administered everyday at once or twice time per a day.
This attributes to the poor stability of G-CSF in blood and short half-life, as well as to the requirement that G-CSF should exist at more than a certain concentration in the blood for keeping its drug efficacy. For this reason, a patient is burdened with everyday administration, and hence burdened with excess use of G-CSF. Therefore, preparation that makes it possible to keep the blood level of G-CSF constant is required.
In view of the fact that granulocyte colony-stimulating factor (G-CSF) forms a precipitate with metal ions such as calcium ion and zinc ion, one conventional art attempted to develop a sustained-release preparation based on such a water-insoluble precipitate. For example, a precipitate formed exclusively of a protein and a multivalent metal ion is easy to dissolve, so that desired sustained releasability cannot be achieved as it is. To cope with this, a method in which a second precipitable substance is added to the precipitate composition formed of G-CSF and metal ion for suppressing dissolution of the precipitate formed of G-CSF and metal ion has been proposed (Japanese Patent Laid-Open Publication No. 2003-81865). In this case, the second precipitable substance to be added is, for example, proteins that can bind with a metal ion but have almost no drug efficacy by themselves, such as human serum albumin and the like. It is also disclosed that by adding an acidic mucopolysaccharide such as chondroitin sulfate, the mixed protein can precipitate more efficiently, and the resultant precipitate shows much better sustained releasability of G-CSF.
The above method, however, entails the problem that although 95% or more of G-CSF precipitates by coprecipitation with these proteins when used at a concentration of around several hundred μg/ml, the precipitation efficiency decreases to 90% or lower when the concentration of G-CSF is 1 mg/ml or more. Additionally, since human serum albumin or chondroitin sulfate used as a coprecipitate is a biological material, there arises a safety problem. Also the above method should counter the problem that the use of the biological materials that are necessarily added is refrained because they are expensive and give not a little influence on the production cost. Furthermore, considering the number of process steps depending on the type of composition, there is still room for improvement.
International Publication Number WO 03/000282 discloses combining a growth hormone, sodium hydrogen carbonate, and zinc acetate for the purpose of solidifying the growth hormone (see Examples 1 and 2), and however, it lacks reference to sustained release effect of the formed solid growth hormone. Also, this differs from the composition found by the present inventors for achieving sustained releasability.
Therefore, it is an object of the present invention to provide a water-insoluble preparation which can be prepared more easily than using the aforementioned methods and capable of precipitating a physiologically active protein or peptide such as G-CSF with high yield and stabilizing the same, and enabling the physiologically active protein or peptide to retain its drug efficacy for several days in a living body owing to the sustained-release effect of the obtained precipitate.
In order to achieve this object, the present inventors examined a method that utilizes co-precipitation with a water-insoluble salt of zinc for finding out a substance which is able to make a physiologically active protein having interaction with a zinc ion precipitate with high efficiency in the presence of a zinc ion without using a biological material such as human serum albumin or chondroitin sulfate as descried above.
As a result, it was found that a physiologically active protein having interaction with a zinc ion is contained efficiently in a precipitate formed by mixing a water-soluble zinc salt such as zinc chloride or zinc acetate with a water-soluble carbonate such as sodium hydrogen carbonate or sodium carbonate and/or a water-soluble phosphate such as sodium phosphate.
In brief, the present inventors found that if a zinc binding physiologically active protein such as G-CSF coexists during formation of a precipitate by mixing a water-soluble zinc salt and an aqueous solution of water-soluble carbonate and/or water-soluble phosphate, a precipitate efficiently containing such a physiologically active protein can be formed. Also it was confirmed that the precipitate composition thus obtained could release the physiologically active protein contained therein in a sustained manner.
Also it was found that by using combination of water-soluble carbonate and water-soluble phosphate and varying the mixing ratio thereof, it is possible to control the sustained-release rate of the physiologically active protein such as G-CSF.
As a particulate sustained-release preparation using a similar water-insoluble inorganic salt, a calcium carbonate preparation disclosed in Japanese Patent Laid-open Publication No. 2002-348234 is known. In this case, it is required to form calcium carbonate particulates at a concentration of 1 M or more for making the resultant precipitate contain G-CSF of about 100 μg/ml. Furthermore, since release amount of G-CSF from the resultant precipitate is very small, and therefore, the characteristic of the precipitate is different from that of the precipitate composition of the present invention.
For example, according to the present invention, almost all G-CSF of approximately 1 mg/ml of G-CSF in concentration can be incorporated in the resultant precipitate by simultaneously mixing approximately 20 mM zinc acetate or zinc chloride (zinc concentration: approximately 1.3 mg/ml) and approximately 20 mM sodium hydrogen carbonate and/or a phosphate.
That is, the present inventors found that almost 100% of G-CSF can be precipitated, for example, with zinc of nearly equivalent weight to G-CSF by using a water-soluble zinc salt and a water-soluble carbonate and/or phosphate, and achieved the present invention.
The obtained precipitate is fine enough to pass through an injection needle, so that it can be used as an injectable preparation.
Furthermore, experiment studies using mice have demonstrated that the present invention improves not only yield of precipitate formation of a physiologically active protein but also sustained-release effect in a living body, and the present invention was accomplished.